One of the most important problems yet to be solved in biochemistry concerns the details of how the cell can regulate its own biosynthesis. The understanding of cellular regulation would have a great impact on our understanding of cellular differentiation and as a consequence of this cures for cancer and birth defects may be found. This proposal will concentrate on the molecular details of how the actual protein molecules involved in the regulatory process function. Emphasis will be directed towards the pyrimidine biosynthesis pathway, the products of which are necessary for DNA replication. Of particular concern will be the enzyme aspartate transcarbamylase which exerts allosteric control over the entire pathway. In order to produce detailed information concerning how this enzyme functions on the molecular level, new data will be produced that can be directly correlated with the presently available biochemical and crystallographic data. This will be accomplished by analysis of a series of mutant aspartate transcarbamylase molecules each having a single amino acid different from the normal form of the enzyme. Production of these mutant enzymes was achieved by suppression of a nonsense codon situated in the gene which codes for the enzyme. Biochemical characterization will be performed on those mutants having altered homotropic and/or heterotropic properties. Correlations will be made between the biochemical properties of the mutants and the molecular level perturbations caused by the amino acid substitution in terms of the enzyme's three-dimensional structure. The data obtained from the mutants should yield details of the allosteric control of this pathway which could not be obtained in any other manner. Further biochemical studies are planned with the native form of the enzyme so that more detailed comparisons can be made with the mutants.